Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ

Abstract

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKC␦, we aimed to understand the role of PKC␦ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-␣, IL-1␤, and IFN-␥. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKC␦ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKC␦, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes.Diabetes is characterized by a progressive decrease in function and mass of ␤-cells, which comprise the majority of cells in the islets of Langerhans (1). Pro-inflammatory cytokines have been implicated as mediators of ␤-cell death in both type 1 diabetes (T1D) 2 and type 2 diabetes (T2D) (2-4). However, pro-inflammatory cytokines also play a role in causing ␤-cell dysfunction early in disease progression (3, 5). In T1D, high levels of pro-inflammatory cytokines, including tumor necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-1␤), and interferon ␥ (IFN-␥), are released by immune cells, such as CD4 ϩ and CD8 ϩ T-cells and macrophages, which infiltrate the pancreas (3, 6). In T2D, adipocyte stress resulting from obesity can lead to secretion of low levels of circulating TNF-␣ from activated macrophages in adipose tissue; whereas elevated free fatty acids and/or hyperglycemia can also lead to local release of IL-1␤ in the islets (4, 7). Although the mechanisms of cytokine-induced cell death in diabetes are well characterized, cytokine-induced islet dysfunction is poorly understood.In vitro, the pro-inflammatory cytokines TNF-␣, IL-1␤, and IFN-␥ work synergistically (8) to induce islet dysfunction and disrupt insulin secretion (9, 10). The effect of pro-inflammatory cytokines on the ␤-cell is thought to be mediated in part by...

Exendin‐4 overcomes cytokine‐induced decreases in gap junction coupling via protein kinase A and Epac2 in mouse and human islets

“…As previously reported (Farnsworth et al . ), cytokine treatment decreased the stimulation index (Fig. A , P = 0.036).…”

“…As previously reported (Farnsworth et al . ) cytokine treatment decreased glucose‐stimulated (11 mM glucose) Ca 2+ pulsatility, oscillation coordination ( P = 0.007, Fig. A and B , Fig.…”

“…A ), as previously reported (Farnsworth et al . ). E4 treatment prevented cytokine‐induced decreases in gap junction coupling over the 1 h treatment ( P < 0.001); however, E4 treatment alone for 1 h was not sufficient to increase gap junction coupling (Fig.…”

“…Image analysis was done using ImageJ to quantify the number of nuclei per islet slice by manual counting and a MATLAB program to identify and quantify the number of Cx36 gap junction plaques and total area of Cx36 staining, as previously reported (Farnsworth et al . ). Contiguous regions of Cx36 positive staining were automatically identified and plaques that did not colocalize with membrane NCAM staining were removed, ensuring only Cx36 on the cell surface was included in further analysis.…”

“…), disrupt Cx36 gap junction coupling (Farnsworth et al . ), decrease insulin secretion (Eizirik et al . ; Andersson et al .…”